Está en la página 1de 14

Research

Plant compartment and biogeography affect microbiome


composition in cultivated and native Agave species
Devin Coleman-Derr1,2,3, Damaris Desgarennes4, Citlali Fonseca-Garcia4, Stephen Gross1,2, Scott Clingenpeel1,2,
Tanja Woyke1,2, Gretchen North5, Axel Visel1,2,6, Laila P. Partida-Martinez4 and Susannah G. Tringe1,2
1

Department of Energy,Joint Genome Institute, Walnut Creek, CA 94598, USA; 2Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; 3Plant Gene

Expression Center, USDA-ARS, Albany, CA 94710, USA; 4Departamento de Ingeniera Genetica, Centro de Investigacion y de Estudios Avanzados, Irapuato 36821, Mexico; 5Department of
Biology, Occidental College, Los Angeles, CA 90041, USA; 6School of Natural Sciences, University of California, Merced, CA 95343, USA

Summary
Authors for correspondence:
Axel Visel
Tel: +1 510 495 2301
Email: avisel@lbl.gov
Laila P. Partida-Martinez
Tel: +52 462 623 9658
Email: laila.partida@ira.cinvestav.mx
Susannah G. Tringe
Tel: +1 925 296 5813
Email: sgtringe@lbl.gov
Received: 1 July 2015
Accepted: 31 August 2015

New Phytologist (2015)


doi: 10.1111/nph.13697

 Desert plants are hypothesized to survive the environmental stress inherent to these regions
in part thanks to symbioses with microorganisms, and yet these microbial species, the communities they form, and the forces that influence them are poorly understood.
 Here we report the first comprehensive investigation of the microbial communities associated with species of Agave, which are native to semiarid and arid regions of Central and North
America and are emerging as biofuel feedstocks. We examined prokaryotic and fungal communities in the rhizosphere, phyllosphere, leaf and root endosphere, as well as proximal and
distal soil samples from cultivated and native agaves, through Illumina amplicon sequencing.
 Phylogenetic profiling revealed that the composition of prokaryotic communities was
primarily determined by the plant compartment, whereas the composition of fungal communities was mainly influenced by the biogeography of the host species. Cultivated A. tequilana
exhibited lower levels of prokaryotic diversity compared with native agaves, although no
differences in microbial diversity were found in the endosphere.
 Agaves shared core prokaryotic and fungal taxa known to promote plant growth and
confer tolerance to abiotic stress, which suggests common principles underpinning Agave
microbe interactions.

Key words: Agave, biogeography, cultivation, desert, iTags, microbial diversity, plant
microbiome, plantmicrobe interactions.

Introduction
The genus Agave, native to the deserts and dry lands of central
Mexico and the southwestern USA, includes a number of
promising candidate biofuel crops for arid and semiarid climates (Somerville et al., 2010; Davis et al., 2011). There are
> 200 species of Agave, and several of these have been cultivated for centuries for the production of alcohols and fiber
(Garcia-Moya et al., 2011). Recent research has highlighted the
suitability of agaves as biofuel feedstocks as a consequence of
their comparatively low lignin content, ease of decomposition,
and high yields with minimal resource inputs (Davis et al.,
This manuscript has been authored by an author at Lawrence Berkeley
National Laboratory under Contract no. DE-AC02-05CH11231 with the
US Department of Energy. The US Government retains, and the publisher,
by accepting the article for publication, acknowledges, that the US
Government retains a non-exclusive, paid-up, irrevocable, world-wide license
to publish or reproduce the published form of this manuscript, or allow
others to do so, for US Government purposes.

2014; Li et al., 2014). Additionally, agaves are capable of


growing on otherwise nonarable rocky soils in regions characterized by prolonged drought and extreme temperatures owing
in part to physiological and biochemical adaptations that prevent excess water loss (Davis et al., 2011; Shakeel et al., 2012;
Campos et al., 2014). This ability to grow on subprime land
reduces the competition for valuable and limited arable agricultural land between Agave and other food crops (Borland et al.,
2009; Holtum et al., 2011).
However, the economic viability of Agave-based biofuels will
hinge on the maximization of plant yield (N
un
~ez et al., 2011).
Conventional methods of crop improvement with agaves are possible, but are made more difficult by the long period plants take
to reach maturity; transgenic approaches could accelerate this
process but have been impeded by the limited tools for genetic
transformation of Agave (Flores-Bentez et al., 2007; EscamillaTrevi~
no, 2012).
It is now well established that symbiotic bacteria and fungi
have profound impacts on plant health and adaptation to stress

No claim to US Government works


New Phytologist 2015 New Phytologist Trust
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.

New Phytologist (2015)


www.newphytologist.com

New
Phytologist

2 Research

(Lugtenberg & Kamilova, 2009; Partida-Martnez & Heil, 2011;


Gaiero et al., 2013; Mendes et al., 2013; Philippot et al., 2013;
Panke-Buisse et al., 2015) and that manipulation of the plant
microbiome has the potential to dramatically improve the yield
of agronomically important crops (Turner et al., 2013). Indeed,
it has been proposed that plants should be considered a collective
holobiont rather than standalone entities (Zilber-Rosenberg &
Rosenberg, 2008; Vandenkoornhuyse et al., 2015). Recent
research has demonstrated that plant-growth-promoting
microbes (PGPM) harvested from plants grown in semiarid environments are capable of enhancing plant growth and stress
tolerance in a variety of crop species (Marasco et al., 2012; Mengual et al., 2014; Rolli et al., 2015). In Agave, recent research
highlighted the likely beneficial role of native associated diazotrophic bacteria, which may play a role not only in nutrition,
but also in tolerance to drought (Desgarennes et al., 2014). Also,
previous reports in which either mycorrhizal or endophytic fungi
were found to promote plant growth in Agave species (Cui &
Nobel, 1992; Obledo et al., 2003) suggest that the Agave microbiome plays an important role in the fitness and productivity of
this genus. Thus, an alternative to improve Agaves yield may be
the rational use and manipulation of the microbiome.
Unlike many food crops, current commercially important Agave
species and cultivars have undergone limited domestication efforts,
despite having been grown under continuous cultivation for centuries in plantations that exist in proximity to related native populations (Nobel, 2003). Therefore, the genus also offers an
opportunity to evaluate whether cultivation is associated with
changes in the composition of microbial communities associated
with a plant host, as has been suggested for other species by recent
studies (Edwards et al., 2015). Importantly, studies in other plants
and soil systems have provided divergent results, describing examples of both decreased (Fierer et al., 2013) and increased microbial
diversity (Rodrigues et al., 2013) upon cultivation.
Previous studies investigating the microbial communities associated with diverse plant hosts have demonstrated that sample
type (plant compartment), host species, geographic location, and
season are all capable of influencing community composition,
although the extent to which each plays a role has varied from
study to study (DeAngelis et al., 2008; Lundberg et al., 2012;
Lanconi et al., 2013; Peiffer et al., 2013; Mendes et al., 2014;
Edwards et al., 2015; Zarraonaindia et al., 2015). Most previous
studies have focused on prokaryotic communities, providing limited insights into the forces shaping plant-associated fungal communities. While recent comparative studies (Hilton et al., 2013;
Shakya et al., 2013) indicate that distinct factors can drive fungal
and prokaryotic rhizosphere communities, it is unclear to what
extent these differences extend to other plant compartments,
plant hosts and soil systems.
The primary goal of this research was to investigate the
prokaryotic and fungal communities associated with the bulk and
proximal soil, the rhizosphere, the phyllosphere, and the root and
leaf endospheres, for three Agave species: the cultivated Agave
tequilana and the native species, Agave salmiana and Agave deserti
(Fig. 1ac). These species were selected based on their natural
geographic distribution (Table 1; Fig. 1a) (CONABIO, 2006;
New Phytologist (2015)
www.newphytologist.com

Gentry et al., 2013), their differentiated management status (cultivated versus native), and their relevance to both existing and
prospective industries. Comparisons across these three Agave
species would allow us to infer the impact of biogeography and
cultivation status on microbial composition and diversity in the
genus Agave. Additionally, we also evaluate the major biotic (i.e.
plant species and plant compartment) and abiotic (i.e. season and
site) factors shaping both prokaryotic and fungal communities,
and identify prominent microbial players in order to develop
rational microbiome-based strategies for improving the yield and
productivity of Agave.

Materials and Methods


Experimental design
Our study investigated three Agave species: Agave tequilana
F.A.C. Weber, Agave salmiana Otto ex Salm subsp. crassispina
(Trel.) Gentry, and Agave deserti Engelm. These species are distributed in central Mexico and in southern California, but they
are not sympatric (Fig 1a). Agave tequilana belongs to the group
Rigidae (Gentry, 1982) and exists only in cultivated populations,
mainly in Jalisco (Fig. 1a). Agave salmiana belongs to the group
Salmianae, whose natural populations extend from Sonora to
Guerrero in western Mexico (Gentry, 1982; CONABIO, 2006).
Agave deserti belongs to the group Deserticolae and it is native to
California and can also be found in parts of Arizona and Baja
California (Gentry, 1982). In 2012, samples were collected at
two different times: in late May (the end of the 8 months of low
or null precipitation in Mexico) and in early October (at the end
of the 4 months of the most precipitation in Mexico). Agave
tequilana samples were taken from two fields managed by tequila
companies in Guanajuato and Jalisco, Mexico: Penjamo (Pe) and
Amatitan (Am) (Fig. 1a), while Agave salmiana samples were collected from the natural populations of El Magueyal (Ma) and
San Felipe (SF) in Guanajuato, Mexico. Agave deserti samples
were collected in September and April (in a year with little rainfall) from the natural populations within the Philip L. Boyd
Deep Canyon Desert Reserve in California called Agave Hill
(AH), Boyd Ridge (BR), and Pinyon Flats (PF). Soil characteristics as well as temperature and precipitation data for each site are
presented in Table 1. For each species, and at each location and
time, samples from three healthy plants were collected. Four samples per plant were collected to analyze six different communities
(Fig. 1b): two leaves for leaf endosphere and phyllosphere; root
tissue for root endosphere and rhizosphere; root zone soil, which
was soil loosely attached to or adjacent to the roots, and bulk soil
gathered 1 m away from the selected plant specimen. All samples
were stored at 4C until processing. We analyzed in total 252
samples; 72 from A. tequilana, 72 from A. salmiana and 108 from
A. deserti.
Sample preparation, DNA extraction, PCR, and sequencing
Samples were prepared and total DNA was extracted as previously described (Desgarennes et al., 2014). For amplification of
No claim to US Government works
New Phytologist 2015 New Phytologist Trust

New
Phytologist

Research 3
(a)

(b)

(c)

(d)

(e)

Fig. 1 Experimental design of this study. (a)


Study sites and biogeography of selected
Agave species. (b) The six samples analyzed
from each plant. (c) Pictures of A. tequilana,
A. salmiana and A. deserti. (d, e) Venn
diagrams of shared prokaryotic (red) and
fungal (blue) operational taxonomic units
across groups of sample types and across
Agave species.

the ITS2 and 16S regions, we used the ITS9F/ITS4R and 515F/
816R primer pairs, respectively, and followed the PCR protocol
described in the Supporting Information. For 16S amplification,
peptide nucleic acid (PNA) clamps were used as in Lundberg
et al. (2013), which substantially reduced overall chloroplast and
mitochondrial contamination and increased the proportion of
prokaryotic reads for the root endosphere, leaf endosphere, and
phyllosphere samples. Paired-end 2 9 250 bp sequencing was
performed on an Illumina MiSeq instrument (Illumina Inc., San
Diego, CA, USA) operating with v2 chemistry. All quality
sequences related to this project are available in the NCBI
Sequence Read Archive (SRA) under project IDs SRA211411,
SRA211420, SRA211416, SRA211422 and SRA211408.
Data processing and statistical analyses
The raw Fastq reads were processed using a custom pipeline
developed at the Joint Genome Institute (Tremblay et al., 2015)
(Supporting Information Methods S1). Raw reads were contaminant-filtered, quality trimmed, merged and clustered to produce 25 871 and 40 759 fungal and prokaryotic operational
No claim to US Government works
New Phytologist 2015 New Phytologist Trust

taxonomic units (OTUs), respectively, at 95% and 97% identity using the UPARSE pipeline (Edgar, 2013). Taxonomies
were assigned to each OTU using the RDP Nave Bayesian
Classifier (Wang et al., 2007) with custom reference databases.
OTUs whose RDP classifications did not match their expected
taxonomic kingdoms (Fungi and Bacteria/Archaea, respectively)
were removed. Average read counts varied by sample type for
both data sets, and in particular the aerial endophytic samples
had substantially fewer reads than the other sample types
(Fig. S1). To reduce low-abundance and spurious OTUs, technical reproducibility thresholds determined empirically from
technical replicates as in Lundberg et al. (2013) (Fig. S2) were
set and OTUs kept only if they had at least two reads in at least
five samples (ITS2 data) or at least seven reads in at least five
samples (16S data).
For diversity analyses, all samples were randomly subsampled
(rarefied) to 1000 reads per sample to account for differences in
the number of reads across samples. We calculated the Shannon
diversity (H0 ) index using the package BiodiversityR in R. All
other statistical analyses were performed in R using a variety of
packages and custom scripts using the measurable OTUs, as
New Phytologist (2015)
www.newphytologist.com

New
Phytologist

4 Research
Table 1 Geographic location, annual mean temperature, precipitation and soil characteristics of study sites
Sites

Latitude
Longitude
Altitude (m above sea level)
Annual mean temperature (C)a
Annual precipitation (mm)a
Precipitation during dry season (mm)a
Precipitation during rainy season (mm)a
Soil edaphic factors
Texture
pHb
Organic matter (%)
Nitrogen (lg g1)
Phosphorus (lg g1)
Potassium (lg g1)

Mexico

USA

Guanajuato
Jalisco
Agave tequilana

California
A. salmiana

Amatitan
(Am)

Penjamo
(Pe)

El Magueyal
(Ma)

San
Felipe (SF)

Boyd
Ridge (BR)

Agave
Hill (AH)

Pinyon
Flats (PF)

21.053
103.902
1260
26.4
558
4.2
553.8
Clay loam
6.8
1.1
19.7
3.64
193

20.686
101.875
1714
19
790
147
643
Clay
6.68
0.96
9.14
27.1
482

21.195
100.439
2175
17.9
485
130
355
Sandy loam
5.79
3.97
8.76
18.11
64.35

21.766
100.163
2089
17
204.5
68
136.5
Sandy loam
6.25
0.71
12.65
4.53
251.7

33.714
116.524
452
23.7
136.0
51.7
83.9
Sandy loam
8.02
nd
nd
42.17
155.5

33.665
116.425
814
20.6
183.0
69.4
113.6
Sandy loam
7.48
nd
nd
87
131

33.601
116.595
1225
18.0
238.3
95.5
142.8
Sandy loam
7.95
nd
nd
74
91.33

A. deserti

a
n Nacional del Agua (CONAGUA). Data from sites in California were obtained from DRI Weather
For sites in Mexico, data were provided by Comisio
Station Data Collection (http://www.wrcc.dri.edu/weather/ucde.html). nd, not determined.
b
Statistically significant difference between species after KruskalWallis test (H2,15 = 10.763; P = 0.0046).

greater statistical support was achieved using this data set instead
of the measurable rarefied data set. In brief, Venn diagrams were
plotted with the function venn.diagram using the package
VennDiagram. Distances were calculated using the vegdist
function of the package vegan for BrayCurtis. Nonmetric multidimensional scaling (NMDS) was performed using Mass and
vegan packages. Permutational ANOVAs (PERMANOVAs)
were performed with the function adonis in the package vegan
as described in Desgarennes et al. (2014). The major microbial
player analyses and graphics were performed using the
average relative abundance and relative frequency of each
OTU in each community (i.e. sample) across the three Agave
species.

Results
Microbial community diversity associated with Agave
species
We analyzed the prokaryotic and fungal communities associated with six sample types taken from three Agave species
through Illumina iTag sequencing on the MiSeq platform.
We obtained 35 770 987 and 27 596 665 total high-quality
reads (Fig. S1), which resulted in 3923 and 3173 OTUs after
applying technical reproducibility thresholds for the prokaryotic and fungal data sets (Fig. S2), respectively. The majority
of all prokaryotic and fungal OTUs discovered in the endospheres (leaf and root) in this analysis were also present in
the episphere (rhizosphere and phyllosphere) and surrounding
soils (Fig. 1d). Similarly, the majority of OTUs associated
with the aboveground portions of the plant were also present
New Phytologist (2015)
www.newphytologist.com

in the belowground plant tissues and soil-derived samples,


although there was a considerably larger portion of fungal
OTUs than prokaryotic OTUs (16.8 and 2.2%, respectively)
detected solely in the aboveground tissues (Fig. S3). Intriguingly, the percentage of OTUs shared between sampling sites
in Mexico and California was considerably smaller for fungal
data sets than for the prokaryotic data sets (18.2 and 72.2%,
respectively; Fig. S3), suggesting that fungal communities were
perhaps more shaped by geographic distance. We also determined that A. salmiana shared a larger fraction of prokaryotic
OTUs with the other native species, A. deserti, than with the
other agave collected in Mexico, the cultivated A. tequilana,
while the opposite trend was observed for fungal OTUs
(Fig. 1e).
The levels of microbial diversity differed significantly among
the sample types and across Agave species, except for the root and
leaf endosphere and the prokaryotic bulk soil (Fig. 2a; Table S1).
In general, alpha diversity as measured by the Shannon (H) index
decreased in value from soil to episphere to endosphere (Fig. 2a,
b). Alpha diversity was marginally higher in the root zone soil
than in the bulk soil for both prokaryotic and fungal communities across all three Agave species, suggesting that root exudates or
other plant-associated factors may be responsible for increasing
local microbial diversity. Remarkably, the prokaryotic alpha
diversity associated with both the rhizosphere and phyllosphere
of the cultivated A. tequilana was substantially lower than that of
the two wild agaves, suggesting a loss of natural microbial diversity (Fig. 2a; Table S1). Furthermore, these levels were even lower
than the average values for the respective endophytic compartments of A. tequilana. However, it was surprising that microbial
diversity in the phyllosphere for all three agaves was almost as
No claim to US Government works
New Phytologist 2015 New Phytologist Trust

New
Phytologist

Research 5
(a)

(b)

Fig. 2 Estimated Shannon (H) index in the


(a) prokaryotic and (b) fungal communities
associated with each sample type for the
three Agave species, shown with  SE.
Superscripts (ad) indicate significant
differences in the marked plant compartment
between plant species, while superscripts
(eg) indicate significant differences between
sample types associated with a plant species.
Statistical support is detailed in Supporting
Information Table S1.

high as diversity in the rhizosphere, despite the harsher environmental conditions that prevail for aboveground plant surfaces
than for underground surfaces.
Across all samples, we detected a total of 33 distinct prokaryotic phyla, although just four of these (Proteobacteria,
Actinobacteria, Firmicutes and Bacteroidetes) comprised on average
> 80% of each plant-associated sample type (rhizosphere, root
endosphere, leaf endosphere, and phyllosphere; Fig. S4). The
plant-associated sample types (epispheres and endospheres) were
enriched for Proteobacteria (KruskalWallis v2 = 50.84;
P < 1.00 9 1012)
and
Actinobacteria
(KruskalWallis
v2 = 11.07; P < 8.790 9 104) and depleted for Acidobacteria
(KruskalWallis v2 = 125.51; P < 2.20 9 1016) with respect to
the surrounding soils, consistent with previous observations for
Agave and other plants (Bulgarelli et al., 2012; Lundberg et al.,
2012; Shakya et al., 2013; Desgarennes et al., 2014). While the
prokaryotic relative abundance profile seemed to be similar in the
bulk and root zone soil samples, community composition differed substantially between the rhizosphere, root endosphere, leaf
endosphere, and phyllosphere (Fig. 3). By contrast, the transition
in relative abundance profiles between sample types for fungal
communities was more gradual than discrete (Fig. 3), with several
lineages exhibiting proportional increases or decreases in abundance across bulk soil, root zone, rhizosphere and root endosphere compartments. Fungal communities across all six sample
types were dominated by the phylum Ascomycota (average 91.2%
of total relative abundance), while Basidiomycota represented a
much smaller portion of the communities (7.7%) (Fig. S4).
Strikingly, very few members of the known arbuscular
No claim to US Government works
New Phytologist 2015 New Phytologist Trust

mycorrhizal fungi (AMF) phylum Glomeromycota (36 OTUs;


1.1%) were detected in the soil, root zone soil, rhizosphere and
root endosphere of the native agaves. Those that were detected
were members of Entrophospora and Glomus spp., and were predominantly associated with the two wild agaves (KruskalWallis
v2 = 23.79; P < 1.01 9 106). Mycorrhizal species were virtually
absent from all samples from A. tequilana, including their associated soils.
We observed a number of differences in prokaryotic and fungal relative abundance patterns between the three Agave species.
For example, in both A. tequilana and A. deserti, the fungal orders
Pleosporales and Eurotiales decreased and increased in average relative abundance, respectively, across the transition from bulk soil
to root endosphere (Fig. 3b,f). In A. salmiana, however, relative
abundances of both these taxonomic groups were relatively stable
between these sample types. By contrast, the fungal order
Capnodiales represented a significant portion of the aerial microbiomes of both Mexican agaves (Fig. 3b,d), but was nearly absent
from all sample types in A. deserti (KruskalWallis v2 = 28.64;
P < 8.70 9 107). Similarly, while the prokaryotic order
Bacillales represented a substantial portion (> 20%) of the leaf
endosphere of both Mexican agaves, it represented < 5% of the
leaf endosphere community for A. deserti (KruskalWallis
v2 = 12.65; P < 3.77 9 104).
The rhizospheres of the cultivated A. tequilana were enriched
for the bacterial orders Pseudomonadales (KruskalWallis
v2 = 6.61; P < 9.97 9 103) and Enterobacteriales (KruskalWallis v2 = 21.37; P < 3.79 9 106) with respect to the native rhizospheres (Figs 3a, S5). Combined, the two orders comprised as
New Phytologist (2015)
www.newphytologist.com

New
Phytologist

6 Research

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 3 Order-level relative abundance plots


of prokaryotic (a, c, e) and fungal (b, d, f)
communities by sample type for A. tequilana
(a, b), A. salmiana (c, d), and A. deserti (e, f).
Asterisks in the legend indicate taxonomic
bins containing operational taxonomic units
that could not be resolved to the order level.

much as 92% of a single A. tequilana rhizosphere sample, and on


average represented 66% of the rhizosphere communities of the
cultivated plants. By comparison, the rhizospheres of the wild
A. deserti and A. salmiana showed considerably more diversity,
with the most abundant orders Actinomycetales and Bacillales representing on average only 14 and 10%, respectively, of the native
rhizosphere communities. The prokaryotic communities of the
bulk soil were more similar across all sampling locations, and
Enterobacteriales or Pseudomonadales comprised < 1% of total
community abundance in all cases. Interestingly, the phyllosphere of the cultivated agave had high average levels of the bacterial orders Pseudomonadales, Enterobacteriales and also
Flavobacteriales (Fig. 3a), which together comprised on average
58% of the phyllosphere community. The abundance of these
few lineages in the epispheres of the cultivated A. tequilana came
at the expense of many other depleted orders found to be significantly more abundant in the native rhizospheres, including
Pirellulales (KruskalWallis v2 = 8.19; P < 4.20 9 103),
Solibacterales (KruskalWallis v2 = 8.23; P < 4.05 9 103),
Acidimicrobiales (KruskalWallis v2 = 12.85; P < 3.39 9 104)
and Rhizobiales (KruskalWallis v2 = 13.25; P < 2.73 9 104).
New Phytologist (2015)
www.newphytologist.com

Factors driving microbial communities in Agave at the


global scale
PERMANOVAs of both prokaryotic and fungal communities
evaluating all the factors considered in the experimental design
and their interactions revealed that sample type, the biogeography of the host plant species and the interaction of these two factors had the most influence on microbial communities, although
in the prokaryotic communities sample type accounted for most
of the variance (63%; Table 2), while in fungal communities the
biogeography of the host plant species played the largest role
(52.7% of variance; Table 2). Season and the interaction between
sample type and season also had significant, albeit small, influences on the fungal communities, which were not observed at the
global scale in the prokaryotic assemblages. It is worth remembering that in these global analyses the factor site could not be evaluated independently of plant species, as the Agave species used in
this study do not grow in sympatry. PERMANOVA results also
confirmed that variation among the three independent replicate
samples of the same sampling time/condition in each species was
generally low (small residuals in each F test; data not shown).
No claim to US Government works
New Phytologist 2015 New Phytologist Trust

New
Phytologist

Research 7

Table 2 PERMANOVA analyses of the microbial communities associated with Agave species considering all factors and their interactions (only significant
factors are displayed; P 0.05)
Prokaryotes
Factora

R2

Global
Sample type5,199
Species2,199
Sample type: species10,199

183.247
107.096
10.517

0.631
0.148
0.072

Bulk soil and root zone soil


Species2,71

88.056

Rhizosphere and phyllosphere


Species2,70
Sample type1,70
Sample type: species2,70
Root and leaf endosphere
Sample type1,58
Species2,58
Sample type: species2,58
Species: season1,58
Season2,58
Sample type: season1,58
a

Fungi
Factora

R2

0.001
0.001
0.001

Species2,190
Sample type5,190
Sample type: species10,190
Sample type: season5,190
Season1,190

350.97
49.04
16.83
1.94
2.54

0.527
0.184
0.126
0.007
0.002

0.001
0.001
0.001
0.016
0.050

0.667

0.001

Species2,71
Sample type1,71

190.848
4.284

0.820
0.009

0.001
0.019

88.016
32.056
7.142

0.591
0.108
0.048

0.001
0.001
0.001

Species2,70
Sample type: species2,70
Sample type1,70
Season1,70

254.033
34.259
56.940
2.985

0.709
0.096
0.080
0.004

0.001
0.001
0.001
0.049

110.965
12.546
7.274
5.228
4.624
3.364

0.479
0.108
0.063
0.045
0.020
0.014

0.001
0.001
0.001
0.001
0.014
0.027

Sample type1,49
Species2,49
Sample type: species2,49
Season1,49
Sample type: season1,49
Sample type: species: season1,49

57.269
17.771
4.451
3.756
3.513
2.617

0.351
0.218
0.055
0.023
0.022
0.016

0.001
0.001
0.001
0.008
0.014
0.049

Subscript numbers indicate the degrees of freedom and residuals of each F test.

PERMANOVA results were corroborated by NMDS plots


using the BrayCurtis distance, where both the prokaryotic and
fungal data sets displayed clustering by sample type, geography
and host species, but the relative contributions of each factor differed across the two data sets (Fig. 4). Similar patterns of clustering were observable for the 16S data set using unweighted
UniFrac distances (Fig. S6); weighted UniFrac distances exhibited less distinct clustering by any of the factors. NMDS ordination for the ITS data set was not performed with UniFrac
because of known issues with generating accurate phylogenetic
trees from the hypervariable ITS2 sequence data (Lindahl et al.,
2013). In the case of the prokaryotic communities, the clusters
that formed corresponded mainly to the different sample types,
starting from the soils at one end (soil and root zone soil), followed by the rhizosphere and phyllosphere, and the root and leaf
endospheres at the far end (Fig. 4a). The NMDS plots revealed
that endosphere samples exhibited the greatest between-sample
variation (Fig. 4a,b), while soil samples clustered more closely
together. Similarly, variation in levels of alpha diversity for endospheres in the prokaryotic communities was larger than for soil
samples (Fig. 2a). The prokaryotic samples formed subclusters
within each sample type based on the biogeography of each host
species, confirming the PERMANOVA results shown in Table 2.
In the case of the fungal communities, the global NMDS plot
(Fig. 4b) revealed that there was a clear distinction between samples from California (left) and Mexico (right), and that soils clustered close to the rhizosphere and phyllosphere, while endophytic
communities were grouped separately from them (Fig. 4b). As
sample type was a major factor contributing to data clustering,
we evaluated the impact of all factors in the epiphytic (Fig. 4c,d)
and endophytic communities (Fig. 4e,f) separately. While both
No claim to US Government works
New Phytologist 2015 New Phytologist Trust

prokaryotic and fungal epiphytic data sets were mainly influenced


by the biogeography of the Agave species, the influence of this
factor was greater in the fungal than in the prokaryotic communities. This trend is visually supported by the NMDS and numerically reflected by the PERMANOVA results (Table 2). In the
episphere, sample type and the interaction sample typespecies
explained together 15.6 and 17.6% of the variance in prokaryotes
and fungi, respectively. Season played only a minor role in the
epiphytic fungal communities (Table 2). Statistical analyses of
the endosphere of agaves suggested that these prokaryotic and
fungal communities were influenced not only by the sample type,
plant species and their interaction (which accounted for 65 and
62.4% of the total variance in prokaryotes and fungi, respectively), but also by the factor season and the interaction of this
factor with the others, which explained 7.9% of the variance in
prokaryotes and 6.1% in fungi (Fig. 4e,f; Table 2). NMDS
results also confirmed that the three independent samples taken
at each sampling time/condition generally clustered close to each
other, as depicted in Fig. S7(af), indicating that intrasample
variation between replicates was generally low.
Factors driving microbial communities in Agave at
the species scale
We were also able to investigate the effect of all factors and their
interactions, including the effect of geographic location, within
each Agave species, as our experimental design considered at least
two locations for each species (two 240 km apart for A. tequilana,
two 130 km apart for A. salmiana, and three within 50 km for
A. deserti; Table 1). From these analyses, in which we performed
statistical tests similar to those described above (Fig. S5; Tables
New Phytologist (2015)
www.newphytologist.com

New
Phytologist

0.4

(b)

Coordinate 2
0.4 0.2 0.0 0.2 0.4

0.0
-0.2
0.6 0.4

Coordinate 2

0.2

(a)

0.6

8 Research

0.5

0.0

0.5

1.0

0.4

0.2

0.0

Coordinate 1

0.2

0.0

0.5

0.0

Coordinate 2
0.5

0.6 0.4 0.2

0.4
0.2
0.4 0.2 0.0

Coordinate 2

0.4

(d)
0.6

(c)

1.0

0.6

0.4

0.2

Coordinate 1

0.0

0.2

0.4

Coordinate 1

Coordinate 2
0.5

0.0

0.6 0.4 0.2 0.0 0.2 0.4 0.6

(f)
0.6 0.4 0.2 0.0 0.2 0.4 0.6

(e)

Coordinate 2

0.2

Coordinate 1

0.6

0.5

0.4

Coordinate 1

Species

Bulk Soi l

0.2

0.0

0.2

0.4

0.6

Coordinate 1

Root Zon e

Rhizosphere

Root

Leaf

Endosphere

Endosphere

Phyllosphere

A. tequilana
A. salmiana
A. deserti
Fig. 4 Nonmetric multidimensional scaling (NMDS) plots for BrayCurtis distances of prokaryotic and fungal communities associated with Agave species.
(a, b) All six sample types; (c, d) only rhizospheres and phyllospheres; (e, f) only root and leaf endospheres.

S1S3), we are able to conclude that the microbial communities


associated with the two native agaves behaved similarly for the
factors evaluated. In both native species, sample type explained
most of the variance across samples, although its influence was
greater in the prokaryotic communities than in the fungal ones
(8581% in prokaryotes; 7462% in fungi for A. salmiana and
A. deserti, respectively). Site played a minor role in the prokaryotic communities of A. salmiana (2.2%; Table S2) and none in
those of A. deserti (Table S3). Fungal communities of A. deserti
were more influenced by the site and the interaction of site with
the sample type (explaining together 15% of the variance), while
in A. salmiana the factor site only accounted for 1.5% of the variance in the fungal data set. By contrast, in the cultivated agave
New Phytologist (2015)
www.newphytologist.com

the factors site and season did influence both prokaryotic and
fungal communities (Table S1), suggesting that abiotic factors
played a more substantial role in shaping the microbiome of
A. tequilana.
Major microbial players associated with Agave
the endosphere
Inspired by the possibility of approaching ecological relationships
as microbial markets (Werner et al., 2012), we made use of the
Pareto concept (the 8020 rule) to identify those prokaryotic and
fungal taxa accounting for 80% of Agaves microbial communities. After their identification, we plotted their average relative
No claim to US Government works
New Phytologist 2015 New Phytologist Trust

New
Phytologist

Research 9

(a)

(b)

Fig. 5 Relative frequency versus relative abundance of major operational taxonomic unit (OTU) players associated with the root endosphere of agaves for
(a) prokaryotes and (b) fungi. Arrows indicate most abundant genera in each case. The number of OTUs and the number of samples (n) are indicated in
the bottom right corner of each plot.

abundance and frequency across each sample type in each Agave


species to infer functional relationships and ecological relevance
(Figs S8S13).
In general, our analyses confirmed low levels of microbial
diversity associated with cultivated A. tequilana, as we identified a
reduced number of microbial taxa playing a major role in this
agave in comparison with the native species (Figs 5, S8, S11).
Despite this reduction, our analyses also revealed that there were
a number of shared major players at the phylum/class level across
the three Agave species studied. At the taxonomic level of individual OTUs, we observed different microbes playing major roles
between native and cultivated agaves.
We found that A. tequilanas prokaryotic leaf endosphere was
dominated by OTUs identified as Acinetobacter and Bacillus
(12% each), while Leclercia (830%) was the dominant OTU for
this compartment in the native agaves (Figs S8S10). We
observed that in A. tequilanas root endosphere the major players
were OTUs identified as Stenotrophomonas (33%) and
Agrobacterium (17%) (both Proteobacteria). By contrast, the root
endosphere of native agaves was dominated by
Actinosynnemataceae and Promicromonospora (16% for each, both
belonging to Actinobacteria), plus Rhizobiales (11%) in
A. salmiana and Leclercia (15%) in A. deserti (Fig. 6a). Notably,
the differences observed in the major bacterial players in the
endophytic compartment were not evident in the soil communities across the three plant species (Figs S8S10).
In the fungal communities, we found that in the leaf endosphere of A. tequilana and A. salmiana three of the most abundant OTUs belonged to Alternaria (9 and 14%, respectively),
Penicillium (7% in A. tequilana) and Cladosporium (20% in
No claim to US Government works
New Phytologist 2015 New Phytologist Trust

A. salmiana) (Figs S11, S13). By contrast, in A. desertis leaf


endosphere the dominant OTUs were identified as Psathyrella
(17%) and Cyphellophora (13%) (Fig. S13). Likewise, we
observed that native agaves root endospheres were dominated by
Lophiostoma (611%), plus Cladosporium (6% in A. salmiana)
and Cyphellophora (11% in A. deserti), while A. tequilanas root
endosphere was dominated by Penicillium (28%) and Fusarium
(11%) (Fig. 5b). Finally, we observed that the number of fungal
OTUs and taxa detected in the proximal and distal soil communities in the native agaves was greater than those observed in
A. tequilana (Figs S9S11).
Is there a dry core endophytic microbiome in Agave?
Our experimental design allowed us to evaluate the impact of
naturally occurring dry periods on the microbial communities
associated with Agave. Our Agave samples from Mexico were collected at the highest peak of drought (dry season) and after
4 months of variable levels of precipitation (rainy season)
(Table 1). Based on results from NMDS and PERMANOVA,
which suggested that season influenced prokaryotic and fungal
endophytic communities (Fig. 3e,f; Table 2), we investigated
whether root and leaf endophytic major microbial players were
affected by these seasonal changes (Fig. S14; Tables S4, S5). In
the case of the endophytic prokaryotic communities, we observed
that a core group formed by OTUs of Actinobacteria, Bacilli,
Alpha-, Beta- and Gammaproteobacteria was enriched during the
dry season in comparison to the rainy season for leaf endospheres
of A. tequilana and A. salmiana, as well as for the root endosphere
of A. tequilana, but not in either endosphere of A. deserti (Figs 6a,b,
New Phytologist (2015)
www.newphytologist.com

New
Phytologist

10 Research
(a)
As.Ma As.Sf At.Am At.Pe

Dry

Leaf
endosphere
Root
endosphere

Rainy

Leaf
endosphere
Root
endosphere

(b)

Bacterial Taxa
Crenarchaeota
Acidobacteria
Actinobacteria
Flavobacteria
Sphingobacteria
Chloroflexi
Bacilli
Clostridia
Gemmatimonadetes
Nitrospirae
Planctomycetes
Alphaproteobacteria
Betaproteobacteria
Deltaproteobacteria
Gammaproteobacteria
Verrucomicrobia

S14; Tables S4, S5). This may be explained by the fact that
A. deserti occupies the driest habitat of the three species examined
(Nobel, 2003), experiencing smaller seasonal variations.
In contrast to these differences observed for prokaryotic communities, fungal endophytic communities were less affected by
season (Fig. S15; Table S5). At higher taxonomic levels, the leaf
endospheres associated with A. tequilana and A. salmiana were
similar to one another across both dry and rainy seasons
(Fig. S15). In general, while fungal root endosphere communities
of the three Agave species could be differentiated from one
another (Fig. 4f) based on total relative abundance patterns, they
did in fact share a core of eighty OTUs representing c. 40% or
more of the total community in each species (Figs S14, S15).

Discussion
Distinct factors shape prokaryotic and fungal communities
in Agave
The microbial communities associated with plant hosts are probably shaped by a wide variety of environmental and host-related
factors, including geographic location, plant phenotype and
genotype, soil chemistry, and seasonal effects. This study determined that prokaryotic communities associated with agaves were
chiefly influenced by the sample type or plant compartment,
where the rhizosphere, the phyllosphere and the root and leaf
endosphere were clearly distinct from one another and also from
the surrounding soils. These results were previously suggested for
agaves (Desgarennes et al., 2014), but by using next-generation
sequencing (NGS), these differences were made more clear even
New Phytologist (2015)
www.newphytologist.com

Fig. 6 Endophytic core of agaves. (a)


Nonmetric multidimensional scaling (NMDS)
plot of the endophytic prokaryotic
communities from Agave tequilana and
A. salmiana; grey ellipses note dry season
samples. As.Ma, A. salmiana from site El
Magyuel; As.Sf, A. salmiana from San Felipe;
At.Am, A. tequilana from Amatitan; At.Pe,
A. tequilana from Penjamo. (b) Shared core
bacterial operational taxonomic units
(belonging to Actinobacteria, Bacilli, and
Alpha-, Beta- and Gammaproteobacteria) in
the root and leaf endosphere of agaves in the
dry and rainy seasons. KruskalWallis test
(leaf: v2 = 7.5; df = 1; P-value = 0.00617
for A. tequilana; v2 = 4.5; df = 1;
P-value = 0.03389 for A. salmiana; root:
v2 = 8.31; df = 1, P-value = 0.0039 for
A. tequilana). Relative abundance profiles of
the core prokaryotic taxa, indicated in the
legend  SD for the total relative abundance,
are displayed.

among epiphytic groups. Moreover, by including a Californian


species, A. deserti, living > 2000 km apart from A. tequilana and
A. salmiana, our data revealed the convergence of prokaryotic
communities in Agave, regardless of cultivation status and biogeography. Interestingly, fungal communities presented a different pattern, in which the biogeography of the plant host species
played the dominant role. Recent studies in several different
experimental systems have similarly reported that fungal communities appear to be differentiated more by geographic distance
than are prokaryotic communities (Peay et al., 2007; Shakya
et al., 2013; Meiser et al., 2014), which suggests that fungal
endemism may be a community-shaping force operating at multiple scales and in multiple habitats. One hypothesis suggests that
dispersal limitation is the root cause of this phenomenon (Taylor
et al., 2006), and that fungi behave more like plants and animals
than do bacteria in this respect. Our analyses and comparisons
across sampling sites within individual species substantiate the
suggestion that geography plays a larger role in driving fungal
than prokaryotic communities. The considerably smaller proportion of shared fungal OTUs than prokaryotic OTUs between
samples collected in California and Mexico lends further support
to this hypothesis. Nevertheless, the six microhabitats or sample
types investigated represent, in both prokaryotic and fungal data
sets, a source of major selection. Prokaryotic and fungal epiphytic
communities in Agave revealed a strong influence of the plant
species biogeography (Fig. 4c,d), which can be related to a plant
selection and/or niche effect as previously discussed (Desgarennes
et al., 2014; Mendes et al., 2014). To what extent this phenomenon depends not only on the biogeography of the plant
host species, but also on the host genotype must still be
No claim to US Government works
New Phytologist 2015 New Phytologist Trust

New
Phytologist
investigated by comparing microbial communities of agaves with
those associated with other sympatric Agave or non-Agave species
in these arid environments. Recent studies have demonstrated
that plant host-specific traits, including broad morphological
characteristics (Kembel et al., 2011) and specific genetic pathways
and gene products (Horton et al., 2014; Lebeis et al., 2015), can
have significant effects on microbiome composition and diversity.
In the endosphere, an influence of the season was observed in the
Mexican agaves (Figs 6, S13; Table S4), especially in the prokaryotic communities, as detected previously (Desgarennes et al.,
2014). Leaf endophytic fungal communities were similar in
A. tequilana and A. salmiana, which suggests common mechanisms of plantfungus interaction in this habitat.
Low levels of microbial diversity in the cultivated
A. tequilana compared with native agaves
Cultivated agaves are susceptible to a number of diseases, many
of which cause significant losses in yield and revenue each year
(Dalton, 2005). One of the most costly of these diseases, called
soft rot, was previously estimated to have cost the tequila industry $200 million yr1 (Jimenez-Hidalgo et al., 2004). A putative
causative agent of soft rot has been identified as a member of
the bacterial family Enterobacteriaceae (Pantoea agglomerans)
(Jimenez-Hidalgo et al., 2004). In this study, we observed significantly lower levels of prokaryotic diversity in the rhizosphere and
phyllosphere of the cultivated A. tequilana compared with the
native A. salmiana and A. deserti. Furthermore, this lower alpha
diversity was the direct result of the dominance in the epispheres
of A. tequilana of a few bacterial families, including genera
belonging to Enterobacteriaceae (Pantoea, Leclercia, and
Trabusiella). The lack of enrichment for Enterobacteriaceae in the
surrounding bulk soils suggests that these bacterial species have
adapted to specific associations with their plant host. A number
of agronomic and regulatory practices within the tequila industry
may be potentially influencing prokaryotic alpha diversity in
A. tequilana. First, continuous monocultures, as is typically the
case on agave plantations, have been shown to decrease microbial
community diversity (Hilton et al., 2013). Second, tequila plantations typically grow young agave plants indoors for a period of
time before transplanting to the field, and, before transplant, the
roots are sterilized in formaldehyde and left to dry for a period of
time (Davis et al., 2011). As rhizosphere microbial communities
have been shown to assemble very early in plant development
(Chaparro et al., 2014), root sterilization probably alters the progression and development of a natural microbial community.
Third, governmental regulations that limit the available genetic
pool and propagation techniques (vegetative reproduction via
bulbils and rhizome offshoots preferred) result in agave plantlets
that are genetically identical to the parental line (Valenzuela,
2011). High genetic homogeneity of the A. tequilana cultivar
used in the tequila industry may have allowed the pathogenic
microbes to evolve strategies to evade the limited arsenal of available host defenses. Current research aimed at adapting agave as a
biofuels feedstock is exploring the use of a variety of Agave
species, including the main cultivars from both the fiber and
No claim to US Government works
New Phytologist 2015 New Phytologist Trust

Research 11

alcohol industries as well as traditionally uncultivated species


(Garcia-Moya et al., 2011; Escamilla-Trevi~
no, 2012; Li et al.,
2014), thereby potentially increasing the cultivated agaves
resilience to pathogen attack by introducing additional defensive
capabilities into the collective gene pool. Moreover, vegetative
reproduction of Agave species either in agricultural settings or in
natural environments may also have an influence on the endophytic communities, as microorganisms residing inside the
mother plant could be potentially inherited by the offshoots and
remain present in internal tissues despite surface sterilization.
Our observation that endosphere samples exhibited greater
between-sample variability, compared with episphere and soil
samples, could be explained in part by a combination of the
stochastic nature of endosphere colonization and this cross-generational propagation of endophytic microbes. Further research is
required to evaluate the heritability of the plant microbiome both
in the episphere and in the endosphere compartments of agaves.
Major microbial players highlight convergence across the
genus Agave
In accordance with our global NMDS and PERMANOVA
analyses, an investigation of the major microbial players demonstrated that the prokaryotic and fungal epiphytic and endophytic communities had a reduced number of OTUs playing a
major role in cultivated A. tequilana in comparison with the
native species A. salmiana and A. deserti. This analysis also
revealed that a group of prokaryotic and fungal taxa was conserved across the three Agave species, although the relative distribution of the common taxa varied across the three hosts.
This result is consistent with findings from recent studies in
which the microbiomes of closely related species or cultivars
exhibit both specific microbial lineages with host-specific abundance patterns and a conserved core microbiome (Schlaeppi
et al., 2014; Bulgarelli et al., 2015; Haney et al., 2015; Lebeis
et al., 2015). The variation we observed can be explained by the
fact that these agaves are native to different habitats and represent different genotypes, as microbial communities are assembled on niche-based processes, as a result of the plant selection
effect and environmental factors (Mendes et al., 2013). Interestingly, we found that differences in bulk soil prokaryotic communities seemed not to be significantly correlated with species
management status. By contrast, we found a reduced number of
major fungal players associated with A. tequilana soils in comparison with those of native agaves.
Interestingly, prokaryotic communities in the desert environments surveyed here do not differ markedly at the phylum level
from other soil environments described previously in other phytobiome studies (Lundberg et al., 2012; Bulgarelli et al., 2015;
Edwards et al., 2015; Zarraonaindia et al., 2015). Similarly, the
increased abundance of Proteobacteria and decreased presence of
Acidobacteria in the plant-associated samples with respect to the
surrounding soil has been observed for other plant hosts (Edwards et al., 2015; Zarraonaindia et al., 2015). Taken together,
these findings suggest the presence of a set of conserved forces
acting to shape the structure of both the plant-associated and soil
New Phytologist (2015)
www.newphytologist.com

New
Phytologist

12 Research

prokaryotic microbiomes across a wide array of environments


and host species. For fungi, however, most temperate forests are
dominated by Basidiomycota (OBrien et al., 2005); the fungal
communities associated with agaves described here were dominated by members of Ascomycota. This is consistent with studies
of other semiarid plants such as perennial grasses, which are also
predominantly colonized by Ascomycota (Porras-Alfaro et al.,
2011), and is also consistent with the trend we observed within
our study that differences in fungal communities are correlated
with differences in geographic distance.
The presence of Glomeromycotan sequences in our study was
low, but we detected a preferential association of the
Entrophospora genus with A. salmiana and Glomus with A. deserti.
This apparent lack of Glomeromycota may in part be attributable
to primer bias, as it is well known that universal fungal primers
often fail to amplify these basal organisms (Martin & Rygiewicz,
2005). Ongoing metagenomic studies on the rhizosphere of
agaves will help elucidate whether members of the Glomeromycota
were underrepresented in this study.
The endophytic core of Agave
Our extensive analyses on the Agave-associated microbial communities suggested that the endospheres were less influenced by
the biogeography of the host species, but more by the plant compartment and seasonal changes. We corroborated that indeed
there is a group of prokaryotic organisms living inside agaves
which increased in abundance under natural drought conditions,
as already suggested (Desgarennes et al., 2014). We discovered
that fungal leaf endophytic communities were similar across the
Mexican agaves, and that these fungal taxa were not affected by
season. Importantly, a broader group of taxa were shared across
Agave species in the root endosphere, representing in all cases
> 40% of the community. As previously mentioned, Agave
species reproduce in nature mostly through vegetative offshoots
that remain connected to the mother through their lives (Gentry,
1982), probably enhancing the heritability of the endophytic
microbial symbionts. As recent studies have shown that plant
microbial communities can change significantly across the life
span of a single plant for even annual species (Copeland et al.,
2015), it will be interesting to investigate successional dynamics
in perennial species with asexually reproducing lifestyles. Some of
the microorganisms shared among the three Agave species, among
them several previously reported diazotrophic strains (Desgarennes et al., 2014), have been isolated in our laboratories and will
be sequenced in the near future. These efforts will provide
a genomic baseline to further deepen our understanding of
the complex plantmicrobe interactions in arid and semiarid
ecosystems.

Acknowledgements
We thank Kanwar Singh (Joint Genome Institute) for technical
assistance during library construction, Derek Lundberg (Department of Biology, UNC) for support with the PNA protocol for
16S amplification, Edward Kirton (Joint Genome Institute) for
New Phytologist (2015)
www.newphytologist.com

bioinformatic and analytical support, Susanna Theroux (Joint


Genome Institute) for editorial support in the preparation of the
manuscript, and Carly Phillips and Walter Woodside for assistance in the harvesting of A. deserti samples from the Boyd Deep
Canyon Desert Reserve. This project was supported by the JGI
Community Science Program (CSP); the work conducted by the
US Department of Energy Joint Genome Institute, a DOE
Office of Science User Facility, is supported under contract no.
DE-AC02-05CH11231. L.P.P-M. acknowledges also Consejo
Nacional de Ciencia y Tecnologia in Mexico (CONACyT),
which supported this project with two grants: CB-2010-01151007 and INFR-2012-01-197799. The authors declare no
conflict of interest.

Author contributions
S.G., D.C-D., G.N., T.W., A.V., L.P.P-M. and S.G.T. planned
and designed research; D.C-D., D.D., C.F-G. and S.G. performed experiments; S.G., G.N., D.D., C.F-G. and L.P.P-M.
conducted fieldwork; D.C-D. and S.C. prepared libraries and
processed sequencing data; D.C-D., D.D., C.F-G. and L.P.P-M.
analyzed the data; and D.C-D., D.D., A.V., L.P.P-M. and
S.G.T. wrote the manuscript. All authors read and approved the
final manuscript.

References
Borland AM, Griffiths H, Hartwell J, Smith JAC. 2009. Exploiting the potential
of plants with crassulacean acid metabolism for bioenergy production on
marginal lands. Journal of Experimental Botany 60: 28792896.
Bulgarelli D, Garrido-Oter R, M
unch PC, Weiman A, Droge J, Pan Y, McHardy
AC, Schulze-Lefert P. 2015. Structure and function of the bacterial root
microbiota in wild and domesticated barley. Cell Host & Microbe 17: 392403.
Bulgarelli D, Rott M, Schlaeppi K, Loren Ver, van Themaat E, Ahmadinejad N,
Assenza F, Rauf P, Huettel B, Reinhardt R et al. 2012. Revealing structure
and assembly cues for Arabidopsis root-inhabiting bacterial microbiota. Nature
488: 9195.
Campos H, Trejo C, Pe~
na-Valdivia CB, Garca-Nava R, Conde-Martnez FV,
Cruz-Ortega Mdel R. 2014. Photosynthetic acclimation to drought stress in
Agave salmiana Otto ex Salm-Dyck seedlings is largely dependent on thermal
dissipation and enhanced electron flux to photosystem I. Photosynthesis Research
122: 117.
Chaparro JM, Badri DV, Vivanco JM. 2014. Rhizosphere microbiome
assemblage is affected by plant development. ISME Journal 8: 790803.
CONABIO. 2006. Mezcales y diversidad. Mexico City, Mexico: Comision
Nacional para el Conocimiento y Uso de la Biodiversidad.
Copeland JK, Yuan L, Layeghifard M, Wang PW, Guttman DS. 2015. Seasonal
community succession of the phyllosphere microbiome. Molecular PlantMicrobe Interactions 28: 274285.
Cui M, Nobel PS. 1992. Nutrient status, water uptake and gas exchange for three
desert succulents infected with mycorrhizal fungi. New Phytologist 122: 643
649.
Dalton R. 2005. Alcohol and science: saving the agave. Nature 438: 10701071.
Davis SC, Dohleman FG, Long SP. 2011. The global potential for Agave as a
biofuel feedstock. GCB Bioenergy 3: 6878.
Davis SC, LeBauer DS, Long SP. 2014. Light to liquid fuel: theoretical and
realized energy conversion efficiency of plants using Crassulacean Acid
Metabolism (CAM) in arid conditions. Journal of Experimental Botany 65:
34713478.
DeAngelis KM, Brodie EL, DeSantis TZ, Andersen GL, Lindow SE, Firestone
MK. 2008. Selective progressive response of soil microbial community to wild
oat roots. ISME journal 3: 168178.
No claim to US Government works
New Phytologist 2015 New Phytologist Trust

New
Phytologist
Desgarennes D, Garrido E, Torres-Gomez MJ, Pe~
na-Cabriales JJ, PartidaMartinez LP. 2014. Diazotrophic potential among bacterial communities
associated with wild and cultivated Agave species. FEMS Microbiology Ecology
90: 844857.
Edgar RC. 2013. UPARSE: highly accurate OTU sequences from microbial
amplicon reads. Nature Methods 10: 996998.
Edwards J, Johnson C, Santos-Medelln C, Lurie E, Podishetty NK, Bhatnagar
S, Eisen JA, Sundaresan V. 2015. Structure, variation, and assembly of the
root-associated microbiomes of rice. Proceedings of the National Academy of
Sciences, USA 112: 911920.
Escamilla-Trevi~
no LL. 2012. Potential of plants from the genus Agave as
bioenergy crops. BioEnergy Research 5: 19.
Fierer N, Ladau J, Clemente JC, Leff JW, Owens SM, Pollard KS, Knight R,
Gilbert JA, McCulley RL. 2013. Reconstructing the microbial diversity and
function of pre-agricultural tallgrass prairie soils in the United States. Science
342: 621624.
Flores-Bentez S, Jimenez-Bremont JF, Rosales-Mendoza S, Arg
uello-Astorga
 2007. Genetic transformation of
GR, Castillo-Collazo R, Alpuche-Sols AG.
Agave salmiana by Agrobacterium tumefaciens and particle bombardment. Plant
Cell, Tissue and Organ Culture 91: 215224.
Gaiero JR, McCall CA, Thompson KA, Day NJ, Best AS, Dunfield KE. 2013.
Inside the root microbiome: bacterial root endophytes and plant growth
promotion. American Journal of Botany 100: 17381750.
Garcia-Moya E, Romero-Manzanares A, Nobel PS. 2011. Highlights for Agave
productivity.GCB Bioenergy 3: 414.
Gentry H. 1982. Agaves of continental North America. Tuscon, AZ, USA: The
University of Arizona Press.
Gentry LF, Ruffo ML, Below FE. 2013. Identifying factors controlling the
continuous corn yield penalty. Agronomy Journal 105: 295.
Haney CH, Samuel BS, Bush J, Ausubel FM. 2015. Associations with rhizosphere
bacteria can confer an adaptive advantage to plants. Nature Plants 1: 15051.
Hilton S, Bennett AJ, Keane G, Bending GD, Chandler D, Stobart R, Mills P.
2013. Impact of shortened crop rotation of oilseed rape on soil and rhizosphere
microbial diversity in relation to yield decline. PLoS ONE 8: e59859.
Holtum JAM, Chambers D, Morgan T, Tan DKY. 2011. Agave as a biofuel
feedstock in Australia: AGAVE IN AUSTRALIA. GCB Bioenergy 3: 58
67.
Horton MW, Bodenhausen N, Beilsmith K, Meng D, Muegge BD,
Subramanian S, Vetter MM, Vilhja lmsson BJ, Nordborg M, Gordon JI et al.
2014. Genome-wide association study of Arabidopsis thaliana leaf microbial
community. Nature Communications 5: 5320.
Jimenez-Hidalgo I, Virgen-Calleros G, la Vega OM, Vandemark G, OlaldePortugal V. 2004. Identification and characterisation of bacteria causing
soft-rot in Agave tequilana. European Journal of Plant Pathology 110:
317331.
Kembel SW, Eisen JA, Pollard KS, Green JL. 2011. The phylogenetic diversity
of metagenomes. PLoS ONE 6: e23214.
Lanconi MD, Taketani RG, Kavamura VN, Melo IS. 2013. Microbial community
biogeographic patterns in the rhizosphere of two Brazilian semi-arid leguminous
trees. World Journal of Microbiology and Biotechnology 29: 12331241.
Lebeis SL, Paredes SH, Lundberg DS, Breakfield N, Gehring J, McDonald M,
Malfatti S, del Rio TG, Jones CD, Tringe SG et al. 2015. Salicylic acid
modulates colonization of the root microbiome by specific bacterial taxa.
Science 349: 860864.
Li H, Pattathil S, Foston MB, Ding S-Y, Kumar R, Gao X, Mittal A, Yarbrough
JM, Himmel ME, Ragauskas AJ et al. 2014. Agave proves to be a low
recalcitrant lignocellulosic feedstock for biofuels production on semi-arid lands.
Biotechnology for Biofuels 7: 50.
Lindahl BD, Nilsson RH, Tedersoo L, Abarenkov K, Carlsen T, Kjller R,
K~
oljalg U, Pennanen T, Rosendahl S, Stenlid J et al. 2013. Fungal
community analysis by high-throughput sequencing of amplified markers a
users guide. New Phytologist 199: 288299.
Lugtenberg B, Kamilova F. 2009. Plant-growth-promoting rhizobacteria. Annual
Review of Microbiology 63: 541556.
Lundberg DS, Lebeis SL, Paredes SH, Yourstone S, Gehring J, Malfatti S,
Tremblay J, Engelbrektson A, Kunin V, del Rio TG et al. 2012. Defining the
core Arabidopsis thaliana root microbiome. Nature 488: 8690.
No claim to US Government works
New Phytologist 2015 New Phytologist Trust

Research 13
Lundberg DS, Yourstone S, Mieczkowski P, Jones CD, Dangl JL. 2013.
Practical innovations for high-throughput amplicon sequencing. Nature
Methods 10: 9991002.
Marasco R, Rolli E, Ettoumi B, Vigani G, Mapelli F, Borin S, Abou-Hadid AF,
El-Behairy UA, Sorlini C, Cherif A et al. 2012. A drought resistancepromoting microbiome is selected by root system under desert farming. PLoS
ONE 7: e48479.
Martin KJ, Rygiewicz PT. 2005. Fungal-specific PCR primers developed for
analysis of the ITS region of environmental DNA extracts. BMC Microbiology
5: 28.
Meiser A, Ba lint M, Schmitt I. 2014. Meta-analysis of deep-sequenced fungal
communities indicates limited taxon sharing between studies and the presence
of biogeographic patterns. New Phytologist 201: 623635.
Mendes R, Garbeva P, Raaijmakers JM. 2013. The rhizosphere microbiome:
significance of plant beneficial, plant pathogenic, and human pathogenic
microorganisms. FEMS Microbiology Reviews 37: 634663.
Mendes LW, Kuramae EE, Navarrete AA, van Veen JA, Tsai SM. 2014.
Taxonomical and functional microbial community selection in soybean
rhizosphere. ISME Journal 8: 15771587.
Mengual C, Schoebitz M, Azcon R, Rolda n A. 2014. Microbial inoculants and
organic amendment improves plant establishment and soil rehabilitation under
semiarid conditions. Journal of Environmental Management 134: 17.
Nobel PS. 2003. Environmental biology of Agaves and cacti. Cambridge, UK:
Cambridge University Press.
N
un
~ez HM, RodrGuez LF, Khanna M. 2011. Agave for tequila and biofuels:
an economic assessment and potential opportunities. GCB Bioenergy 3: 4357.
Obledo EN, Barraga n-Barraga n LB, Gutierrez-Gonza lez P, Ramrez-Herna ndez
BC, Ramrez JJ, Rodrguez-Garay B. 2003. Increased photosyntethic efficiency
generated by fungal symbiosis in Agave victoria-reginae. Plant Cell, Tissue and
Organ Culture 74: 237241.
OBrien HE, Parrent JL, Jackson JA, Moncalvo J-M, Vilgalys R. 2005. Fungal
community analysis by large-scale sequencing of environmental samples.
Applied and Environmental Microbiology 71: 55445550.
Panke-Buisse K, Poole AC, Goodrich JK, Ley RE, Kao-Kniffin J. 2015.
Selection on soil microbiomes reveals reproducible impacts on plant function.
ISME Journal 9: 980989.
Partida-Martnez LP, Heil M. 2011. The microbe-free plant: fact or artifact?
Frontiers in Plant Science 2: 100.
Peay KG, Bruns TD, Kennedy PG, Bergemann SE, Garbelotto M. 2007. A
strong species area relationship for eukaryotic soil microbes: island size matters
for ectomycorrhizal fungi. Ecology Letters 10: 470480.
Peiffer JA, Spor A, Koren O, Jin Z, Tringe SG, Dangl JL, Buckler ES, Ley RE.
2013. Diversity and heritability of the maize rhizosphere microbiome under
field conditions. Proceedings of the National Academy of Sciences, USA 110:
65486553.
Philippot L, Raaijmakers JM, Lemanceau P, van der Putten WH. 2013. Going
back to the roots: the microbial ecology of the rhizosphere. Nature Reviews
Microbiology 11: 789799.
Porras-Alfaro A, Herrera J, Natvig DO, Lipinski K, Sinsabaugh RL. 2011.
Diversity and distribution of soil fungal communities in a semiarid grassland.
Mycologia 103: 1021.
Rodrigues JLM, Pellizari VH, Mueller R, Baek K, Jesus Eda C, Paula FS, Mirza
B, Hamaoui GS, Tsai SM, Feigl B et al. 2013. Conversion of the Amazon
rainforest to agriculture results in biotic homogenization of soil bacterial
communities. Proceedings of the National Academy of Sciences, USA 110: 988
993.
Rolli E, Marasco R, Vigani G, Ettoumi B, Mapelli F, Deangelis ML, Gandolfi
C, Casati E, Previtali F, Gerbino R et al. 2015. Improved plant resistance to
drought is promoted by the root-associated microbiome as a water stressdependent trait: root bacteria protect plants from drought. Environmental
Microbiology 17: 316331.
Schlaeppi K, Dombrowski N, Oter RG, van Themaat EVL, Schulze-Lefert P.
2014. Quantitative divergence of the bacterial root microbiota in Arabidopsis
thaliana relatives. Proceedings of the National Academy of Sciences, USA 111:
585592.
Shakeel SN, Ul Haq N, Heckathorn S, Luthe DS. 2012. Analysis of gene
sequences indicates that quantity not quality of chloroplast small HSPs
New Phytologist (2015)
www.newphytologist.com

14 Research
improves thermotolerance in C4 and CAM plants. Plant Cell Reports 31:
19431957.
Shakya M, Gottel N, Castro H, Yang ZK, Gunter L, Labbe J, Muchero W,
Bonito G, Vilgalys R, Tuskan G et al. 2013. A multifactor analysis of fungal
and bacterial community structure in the root microbiome of mature Populus
deltoides Trees. PLoS ONE 8: e76382.
Somerville C, Youngs H, Taylor C, Davis SC, Long SP. 2010. Feedstocks for
Lignocellulosic Biofuels. Science 329: 790792.
Taylor JW, Turner E, Townsend JP, Dettman JR, Jacobson D. 2006.
Eukaryotic microbes, species recognition and the geographic limits of species:
examples from the kingdom Fungi. Philosophical Transactions of the Royal
Society B: Biological Sciences 361: 19471963.
Tremblay J, Singh K, Fern A, Kirton ES, He S, Woyke T, Lee J, Chen F, Dangl
JL, Tringe SG. 2015. Primer and platform effects on 16S rRNA tag
sequencing. Evolutionary and Genomic Microbiology 6: 115.
Turner TR, James EK, Poole PS. 2013. The plant microbiome. Genome Biology
14: 209.
Valenzuela A. 2011. A new agenda for blue agave landraces: food, energy and
tequila. GCB Bioenergy 3: 1524.
Vandenkoornhuyse P, Quaiser A, Duhamel M, Le Van A, Dufresne A. 2015.
The importance of the microbiome of the plant holobiont. New Phytologist
206: 11961206.
Wang Q, Garrity GM, Tiedje JM, Cole JR. 2007. Nave Bayesian classifier for
rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied
and Environmental Microbiology 73: 52615267.
Werner JJ, Zhou D, Caporaso JG, Knight R, Angenent LT. 2012. Comparison
of Illumina paired-end and single-direction sequencing for microbial 16S
rRNA gene amplicon surveys. The ISME Journal 6: 12731276.
Zarraonaindia I, Owens SM, Weisenhorn P, West K, Hampton-Marcell J, Lax
S, Bokulich NA, Mills DA, Martin G, Taghavi S et al. 2015. The soil
microbiome influences grapevine-associated microbiota. mBio 6: e02527-14.
Zilber-Rosenberg I, Rosenberg E. 2008. Role of microorganisms in the evolution
of animals and plants: the hologenome theory of evolution. FEMS Microbiology
Reviews 32: 723735.

Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 Raw read counts across all samples grouped by sample
type.
Fig. S2 Established cut-off thresholds for technical reproducibility across OTUs for 16S and ITS2 data sets.
Fig. S3 OTU distributions across sample subsets.
Fig. S4 Phylum-level relative abundance plots of prokaryotic and
fungal communities associated with each Agave species by sample
type.
Fig. S5 Order-level relative abundance plots of prokaryotic communities associated with all rhizosphere and bulk soil samples.
Fig. S6 NMDS clustering for prokaryotic data of all samples
using unweighted and weighted UniFrac distances.

New
Phytologist
Fig. S8 Major prokaryotic taxa associated with Agave tequilana
based on the 8020 rule (Pareto).
Fig. S9 Major prokaryotic taxa associated with Agave salmiana
based on the 8020 rule (Pareto).
Fig. S10 Major prokaryotic taxa associated with Agave deserti
based on the 8020 rule (Pareto).
Fig. S11 Major fungal taxa associated with Agave tequilana based
on the 8020 rule (Pareto).
Fig. S12 Major fungal taxa associated with Agave salmiana based
on the 8020 rule (Pareto).
Fig. S13 Major fungal taxa associated with Agave deserti based on
the 8020 rule (Pareto).
Fig. S14 OTU distributions across Agave species in the endosphere.
Fig. S15 Fungal taxa associated with the endosphere of all three
Agave species and their relative abundance at the dry and rainy
seasons.
Table S1 Estimated Shannon diversity in the prokaryotic and
fungal communities associated with each Agave species
Table S2 PERMANOVA of the microbial communities associated with Agave tequilana considering all factors and their interactions
Table S3 PERMANOVA of the microbial communities associated with Agave salmiana considering all factors and their interactions
Table S4 PERMANOVA of the microbial communities associated with Agave deserti considering all factors and their interactions
Table S5 PERMANOVA of the microbial communities associated with the endosphere of Agave tequilana and A. salmiana considering all factors and their interactions
Methods S1 Methods for sample collection, DNA extraction,
PCR amplification, sequencing, and data processing, including
statistical analyses.
Please note: Wiley Blackwell are not responsible for the content
or functionality of any supporting information supplied by the
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.

Fig. S7 NMDS clustering of all samples by sample type, host


species and location according to the legend.

New Phytologist (2015)


www.newphytologist.com

No claim to US Government works


New Phytologist 2015 New Phytologist Trust

También podría gustarte